SBI4U Culminating Activity Part 1: Genetic Engineering of a Recombinant Plasmid Name:

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1 SBI4U Culminating Activity Part 1: Genetic Engineering of a Recombinant Plasmid Name: Background Read through The Major Steps of Cloning of DNA on page 290 and examine the figure on page 291. This is the process you will be walking through to generate a recombinant plasmid that contains the gene you have been assigned, spliced into pbluescript plasmid vector. You have been given the GI number and a Fingerprint RE for a gene that is linked to a human condition. Your task is determine how to splice this gene into a plasmid to create a recombinant that could be used for purposes. The plasmid you will be using is either pbsks or pbssk. The GI number for pbsks is The GI number for pbssk is Objectives 1. Provide a description of the disease or condition and associated with the gene of interest. 2. Select appropriate enzymes to splice a gene that has been linked to a physical condition into pbluescript plasmid vector. 3. Generate a basic plasmid map of your recombinant. 4. Generate an image of a gel that would be the fingerprint of the recombinant plasmid. Parameters 1. You have access to the following restriction endonucleases, in addition to all the other tools needed for this procedure: AccI, ApaI, BamHI, BstXI, BtgI, EagI, Eco0109I, EcoRI, EcoRV, HincII, HindIII, KpnI, NotI, PvuII, SacI, SacII, SalI, SapI, SmaI, SpeI, XbaI, XhoI, XmaI 2. From the enzymes above, using the NEB Cutter, find one that is upstream of the ORF. If there are more than one that meet this criteria, pick the one that is closest to the ORF but not within it. This will be referred to as RE1 later in this assignment. 3. From the enzymes above, using the NEB Cutter, find one that is downstream of the ORF. If there are more than one that meet this criteria, pick the one that is closest to the ORF but not within it. This will be referred to as RE2 later in this assignment. 4. The Fingerprint RE you have been assigned will cut your gene of interest as well as the plasmid, even after you make your recombinant. 5. Do custom digests of pbsks and pbssk using RE1, RE2 and the Fingerprint RE. Choose the plasmid that results in your gene of interest splicing in line with lacz (i.e. counterclockwise). 6. Provide a proof for the location of the start codon and stop codon using amino acid sequences. You will need to zoom in with the NEB Cutter for this.

2 7. Complete the next page based on your and reasoning. Note whether you are going to use pbluescript KS or SK. Choose your plasmid such that your gene will be in line with the lacz gene (5 à 3 direction of your gene goes counterclockwise). Remember the numbering goes clockwise. Consider whether RE1 will have a higher or lower number than RE2 in your final plasmid. 8. Label the locations of the cut sites on the maps in bp beside their name. 9. Name your recombinant based on the gene name and plasmid used. Put this within the middle of the recombinant plasmid map. Below that, record the total length of the plasmid in bp. 10. Label the recombinant plasmid map showing the two enzymes used to splice the fragments together as well as the locations of the fingerprinting enzyme. Remember to check for the location of the fingerprint enzyme in both the gene of interest as well as the pbluescript. 11. Use one colour for our gene of interest and another colour for the pbs in your recombinant. 12. Generate gel electrophoresis results for your recombinant using the following cuts: a) RE1 b) RE2 c) RE1 & RE2 d) Fingerprint RE 13. Print and attach a copy of the cdna sequence and create a legend and highlight the following parts of the cdna: A) Open Reading Frame from beginning of start codon to end of stop codon in bold B) RE 1 recognition sequence in blue C) RE2 recognition sequence in red D) Fingerprint RE recognition sequence in purple 14. Write a summary describing the disease or condition associated with the gene you have been given. How is the disease diagnosed treated? What is the prognosis for those afflicted with the disease or condition? Describe an example of scientific or medical being done in relation to this gene or the disease. This section is to be no more than two double- spaced pages. Use appropriate sources (databases or online books the NCBI has online books) and cite and reference them according to APA formatting.

3 Recombination Steps 1. Starting DNA and the locations of the RE cut sites. In the blank beside pbluescript in the table below, write KS or SK depending on which plasmid you will use to make your recombinant. Gene Symbol: Gene Name: Plasmid pbluescript Gene ID: cdna length Plasmid length 2961 RE 1 (5 ): cut site # RE 1 (5 ): cut site #: RE 2 (3 ): cut site #: RE 2 (3 ): cut site#: Fingerprint RE: cut site #: Fingerprint RE: cut site #: 2. Maps: Put the sites for the three REs you choose on the cdna map and the plasmid map in their approximate locations relative to each other. Write their position beside their name. Write the fragment lengths within the box and the circle the enzyme cut sites. cdna Map cdna is short for copy DNA. It is a DNA copy of the final mrna transcript, not including the cap and tail. In other words, a DNA version of the mrna transcript after splicing. pbluescript Map

4 Recombinant Plasmid Map RE2:

5 Agarose Gel Electorphoresis of Note: Write the size of the fragment in bp above each band you draw in your gel Lane 1 Lane 2 Lane 3 Lane 4 Lane 5 Lane 6 Lane 1: New England Bioloabs 1 kb DNA Ladder visualized by ethidium bromide staining Lane 2: New England Biolabs 100bp DNA Ladder visualized by ethidium bromide staining

6 SBI4U Genetic Engineering of a Recombinant Plasmid CRITERIA LEVEL 4 LEVEL 3 LEVEL 2 LEVEL 1 < LEVEL 1 Thinking & Investigation - use of critical (e.g. gathering evidence, proving, design & problem- solving) with a high with an adequate with a moderate with a limited with a negligible Communication - use of scientific terminology, symbols, conventions accurately & effectively - expresses ideas clearly shows a high shows a considerable shows a moderate shows a limited shows a negligible Application - making thorough adequate moderate limited negligible